Antifolates are compounds that interfere at various stages of folate metabolism M. G. Nair. "The Chemistry of Antitumor Agents". Chapter, 7; Blackie & Sons. London. 1991!. Folate is a vitamin that is essential for the biosynthesis of purine and pyrimidine nucleotide precursors of DNA. Therefore antifolates are capable of inhibiting DNA biosynthesis and hence cell division. Indeed methotrexate (MTX) which is a powerful antifolate by virtue of its inhibition of Dihydrofolate Reductase (DHFR) mediated production of the active vitamin tetrahydrofolate is curative to Choriocarcinoma and Burkitt's lymphoma. MTX is also widely used as a single agent or in combination with other drugs for the treatment of various forms of human cancers M. G. Nair. "Cancer Growth and Progression: Cancer Management in Man" Volume 10, H. E. Kaiser (Ed); Kluwer Academic Publishers, Chapter, 7, 1989!. The anti-rheumatoid properties of MTX is well documented and it is currently used as an arthritis remittive agent under the trade name Rheumatrex. MTX has also shown activity against asthma, but it has not yet been used clinically for this indication, except on an experimental basis.
In 1973 Nair and Baugh Biochemistry, 12, 3923, 1973 ! discovered that MTX is metabolized to its polyglutamyl derivatives in human and other mammalian tissues. Once formed within the cells, MTX polyglutamates do not readily efflux and they remain for long periods exerting their cytotoxic effects to tumor and normal cells. This prolonged retention of MTX polyglutamates relative to MTX results in the potentiation of host toxicity. In 1991 Nair discovered that U.S. Pat. No. 5,073,554 ! contrary to the widely accepted notion, polyglutamylation of classical antifolates is not essential for anti-tumor activity and in fact this metabolic transformation may indeed cause the loss of pharmacological control and target specificity of the drug. This new finding resulted in the discovery of a number of non-polyglutamylatable classical antifolates and led to the clinical development of methylene-10-deazaaminopterin MDAM! as an experimental anticancer drug for the treatment of human solid tumors Clinical Cancer Research, 2, 707-712, 1996 !. In 1996 Nair and coworkers identified U.S. Pat. No. 5,550,128! the active enantiomer of MDAM as the one that possess the L-configuration L-MDAM!. Further investigation by Nair and coworkers to delineate the metabolic disposition of certain non-polyglutamylatable antifolates led to the unexpected finding that the presence of the 4-methyleneglutamate moiety modulates the binding of such compounds to the liver enzyme aldehyde oxidase which mediates their oxidative deactivation to the corresponding 7-hydroxy derivatives Cellular. Pharmacology, 3, 29, 1996!.
Another in vivo transformation of MTX is the cleavage of the C9-N10 bond during its entry to the enterohepatic circulation. The cleaved products are inactive and therefore constitutes an alternate pathway of metabolic inactivation. It has also been documented that the intestinal microflora are capable of removing the L-glutamate portion of methotrexate and aminopterin to inactive compounds that enter systemic circulation via enterohepatic circulation and further adversily complicate the biochemical pharmacology of these drugs. Taken together, these multiple pathways of drug inactivation and the formation of the 7-hydroxyderivative, its competition with the parent drug for polyglutamylation and transport to target cells and the loss of target specificity secondary to the above metabolic transformation not only undermine the pharmacological control of MTX but also results in loss of efficacy and manifestation of undesirable toxicity.
As part of a continuing program aimed at the development of less toxic and more specific antifolate drugs for the treatment of human cancers, the pteridine ring of the experimental anticancer drug methylene-10-deazaaminopterin MDAM! was replaced with a quinazoline ring. This new compound 1 when evaluated for its potential utility as an antitumor agent using a number of biological test systems exhibited unexpected biological properties. For example 1 was completely inert when incubated with rabbit liver aldehyde oxidase establishing that it is not converted to the corresponding 7-hydroxy derivative. Compounds 1 and 1d on incubation with carboxypeptidase derived from Pseudomonad sp failed to remove the 4-methyleneglutamate moiety establishing that it is resistant to microbial inactivation by intestinal flora. Further, and perhaps due to the total inertness of 1 to metabolic transformation, unlike other classical antifolates 1 was able to kill a large number of human luekemia and human solid tumor cells in culture at therapeutically relevant concentrations. Since compound 1 has a methylene group as opposed to the methylamino group of MTX at the tenth position it is also not subject to bacterial deactivation by cleavage of the bridge bond. When evaluated for inhibitory activity against the growth of a number of human cancer cells in culture, compound 1 was strikingly more active than either MTX or MDAM. Further evaluation in vitro using the enzyme folylpolyglutamate synthetase revealed that 1 and its analogs reported herein are not capable of elaboration to its polyglutamates due to the presence of the 4-methyleneglutamate moiety. There fore the unexpected enhanced biological activity of 1 must have its origin in the complete lack of its in vivo metabolism FIG.-1!. Unlike trimetrexate, the compounds described in this invention are capable of transport to target tumor cells by the reduced folate transporter (RFT) due to the presence of the 4-methyleneglutamate moiety. More strikingly, compound 1 was 4-5 times collaterally more sensitive in inhibiting the growth of leukemia cells that are resistant to MTX by virtue of defective polyglutamylation compared to the wild type MTX sensitive parental CCRF-CEM cell line. Accordingly, this invention demonstrates that metabolically inert classical folate analog inhibitors of dihydrofolate reductase are superior antitumor agents relative to the metabolizable classical antifolates such as methotrexate and aminopterin and to the 7-hydroxylatable but non-polyglutamylatable antifolates MDAM and MEDAM. As a model of metabolically ineffective DHFR inhibitor, compound 1 was evaluated as a potential anti-inflammatory agent relative to MTX and surprisingly it exhibited outstanding activity in this animal model of asthma substantiating the superiority of metabolically inert DHFR inhibitors. In fact 1 was found to be not only superior to MTX but also to the well known anti-asthmatic drug theophylline in both early and late asthmatic responses and bronchial hyper responsiveness (BHR) when evaluated using allergic rabbits. The anticancer and anti-inflammatory antifolates reported in this invention include close analogues of 1 bearing modified C9-10 region incapable of aldehyde oxidase mediated 7-hydroxylation and the 4-methyleneglutamate moiety that prevents polyglutamylation and modulation of binding to aldehyde oxidase. These quinazoline-based compounds 1, 1a, 1b, 1c, and 1d ! may possess the racemic D,L-4-methyleneglutamate, enantiomerically pure D-4-methyleneglutamate "D" configuration ! or enantiomerically pure L-4-methyleneglutamate "L" configuration ! moieties. They are also useful for the treatment of rheumatoid arthritis due to their inhibition of the enzyme dihydrofolate Reductase.
This invention accordingly provides a process for treating neoplastic diseases leukemia, ascetic and solid tumors !, a process for treating asthma and related inflammatory diseases and a process for treating rheumatoid arthritis and other auto immune diseases which comprises administering to a warm blooded animal with an abnormal proportion of leukocytes or other evidence of neoplastic disease, asthma or rheumatoid arthritis a therapeutically effective non-toxic amount of 5,8,10-trideaza-4'-methyleneaminopterin (1) herein referred to as compound 1! or its close analogues 1a-1d as such or in the form of a pharmacologically acceptable salt thereof. They may be combined with other compounds such as leucovorin folinic acid; citrovorum factor! to reduce toxicity or in combination with other anticancer drugs including but not limited to tomudex, 5-FU; 5-FdUR; carboplatin, oxaloplatin or cis-platin; taxol, campothecins or cyclophosphamide to enhance efficacy.
The salts of 1 or its close analogues 1a-1d! may be formed with one or more of the amino groups of the quinazoline ring with acids such as acetic, hydrochloric, sulfuric, sulfonic, nitric, hydrobromic, phosphoric, citric, salicylic or methanesulfonic. Compound 1 or its close analogues and salts thereof may be administered to a warm blooded animal by oral or parenteral (intraperitoneal, intravenous, intrathecal, subcutaneous, intramuscular, etc.) routes. Higher dosage of 1 or its close analogues 1a-1d ! may be administered in conjunction with racemic leucovorin 6-(R,S) 5-formyltetrahydrofolate! and/or folic acid to further reduce toxicity.
Compound 1 or its close analogues 1a-1d ! may be provided in composite forms to facilitate administration to patients or in dosage unit form. A non-toxic and sterile carrier may be added to 1 and its close analogues. This carrier may be solid, liquid or semi-solid that may serve as a medium, vehicle or excipient. Methyl cellulose, polyhydroxybenzoate, talc, gelatin, lactose, dextrose, starch, mannitol, sorbitol, mineral oil, gum acacia, oil of theobroma or magnesium stearate may serve as a carrier. Another useful and preferred formulation of these entities for administration to patients is their conversion to the corresponding sodium or potassium salts by dissolving in either sodium bicarbonate, potassium bicarbonate, sodium carbonate or potassium carbonate solution. The resulting solutions may be used as such or cryodessicated to the solid sodium or potassium salt and conveniently formulated in aqueous or non-aqueous vehicles or carriers. Compound 1, or its close analogues (1a-1d) and a carrier or diluent can be encapsulated or enclosed in a paper or other container, cachet, gelatin, capsule or sachet when intended for use in dosage units.
The process of the invention for the synthesis of compound 1 starts with the conversion of commercially available 5-methyl-2-nitrobenzoic acid to the corresponding amide 2 and its subsequent transformation to 5-methyl-2-nitrobenzonitrile (3) by standard procedures. Reaction of 3 in DMF under nitrogen with p-formylmethylbenzoate in presence of an organic base such as diazabicyclo octane for several hours gave the olefin (4) after work up as a mixture of geometric isomers. Olefin 4 can also be prepared by reacting 3 with p-formylmethylbenzoate in methanol using sodium methoxide as a base. In general this reaction can be performed in any appropriate organic solvents using commonly used organic or inorganic bases. Reduction of 4 with sodium dithionite gave the aminonitrile (5) which was cyclized with guanidine to the corresponding pteroate analogue (6) which after catalytic hydrogenation and hydrolysis gave 4-amino-4-deoxy-5,8,10-trideazapteroic acid (2). Coupling of 7 with diethyl-4-methyleneglutamate by the isobutylchloroformate method previously described by Nair and Baugh Biochemistry, 12,3923-3927, 1973! followed by mild hydrolysis of the resultant diester gave crude 1 which was purified by reverse phase chromatography on C-18 silica gel using 12% acetonitrile in water as the eluting solvent Scheme-1!.
An alternate procedure for the preparation of olefin 4 is allylic bromination of 3 to the corresponding benzyl bromide (3b), and its subsequent reaction with triphenylphosphine to the wittig salt. Treatment of thisWittig salt with p-formylmethylbenzoate in an organic solvent (eg, DMF) using an organic base in a typical Wittig reaction gave 4 in moderate yield. Any covenient organic solvent and an organic or inorganic base compatable with the solvent can be used for this reaction.
Substitution of p-formyl methylbenzoate with p-carbomethoxyacetophenone in the above reaction with 3 gives the corresponding methyl substituted olefin which after dithionite reduction, guanidine cyclization, hydrogenation, hydrolysis, diethyl-4-methyleneglutamate coupling followed by mild hydrolysis yields the 10-methyl derivative 1a. Likewise substitution of p-formyl methylbenzoate with p-carbomethoxypropiophenone in the reaction with 3 and workup as above should yield the 10-ethyl derivative 1b.
Benzylic bromination of 3 gave the corresponding bromomethyl derivative(3b) that on reaction with p-methylaminomethybenzoate and methyl-p-methylaminobenzoate gave the corresponding aminonitriles which after dithionite reduction, guanidine cyclization and hydrolysis gave the pteroate analogs 8 and 9. 4-Methyleneglutamate coupling described as above and hydrolysis gave the 10-nor-methylamino and 10-nor-amino derivatives 1c and 1d respectively.
In order to unravel the mechanism of action of 1 and 1 e they were examined as inhibitors of human dihydrofolate Reductase. Both compounds exhibited inhibitory activity to this enzyme similar to that of MTX. The I.sub.50* values for the human enzyme by 1, 1e and MTX were 66.0, 66.0 and 11.0 nM respectively. The corresponding value for trimetrexate (TMTX) was 54.0 nM.